Lehrich R W, Aller S G, Webster P, Marino C R, Forrest J N
Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
J Clin Invest. 1998 Feb 15;101(4):737-45. doi: 10.1172/JCI803.
Defective trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common cause of cystic fibrosis. In chloride-secreting epithelia, it is well established that CFTR localizes to intracellular organelles and to apical membranes. However, it is controversial whether secretagogues regulate the trafficking of CFTR. To investigate whether acute hormonal stimulation of chloride secretion is coupled to the trafficking of CFTR, we used the intact shark rectal gland, a model tissue in which salt secretion is dynamically regulated and both chloride secretion and cellular CFTR immunofluorescence can be quantified in parallel. In rectal glands perfused under basal conditions without secretagogues, Cl- secretion was 151+/-65 microeq/h/g. Vasoactive intestinal peptide (VIP), forskolin, and genistein led to 10-, 6-, and 4-fold increases in Cl- secretion. In basal glands, quantitative confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell (7.28+/-0.35 micron). During stimulation with secretagogues, apical extension of CFTR immunofluorescence into the cell was reduced significantly to 3.24+/-0.08 micron by VIP, 4.08+/-0.13 by forskolin, and 3.19+/-0.1 by genistein (P < 0.001). Moreover, the peak intensity of CFTR fluorescence shifted towards the apical membrane (peak fluorescence 2.5+/-0.13 micron basal vs. 1.51+/-0.06, 1.77+/-0.1, and 1.38+/-0.05 for VIP, forskolin, and genistein; all P < 0.001). The increase in both Cl- secretion and apical CFTR trafficking reversed to basal values after removal of VIP. These data provide the first quantitative morphological evidence for acute hormonal regulation of CFTR trafficking in an intact epithelial tissue.
囊性纤维化跨膜传导调节因子（CFTR）转运缺陷是囊性纤维化最常见的病因。在分泌氯离子的上皮细胞中，CFTR定位于细胞内细胞器和顶端膜已得到充分证实。然而，促分泌剂是否调节CFTR的转运仍存在争议。为了研究氯离子分泌的急性激素刺激是否与CFTR的转运相关，我们使用了完整的鲨鱼直肠腺，这是一种模型组织，其中盐分泌受到动态调节，并且氯离子分泌和细胞CFTR免疫荧光可以同时进行定量分析。在无促分泌剂的基础条件下灌注的直肠腺中，氯离子分泌为151±65微当量/小时/克。血管活性肠肽（VIP）、福斯可林和金雀异黄素使氯离子分泌分别增加了10倍、6倍和4倍。在基础腺中，定量共聚焦显微镜显示CFTR免疫荧光从顶端膜深入细胞内（7.28±0.35微米）。在用促分泌剂刺激期间，VIP使CFTR免疫荧光向细胞顶端的延伸显著减少至3.24±0.08微米，福斯可林使其减少至4.08±0.13微米，金雀异黄素使其减少至3.19±0.1微米（P<0.001）。此外，CFTR荧光的峰值强度向顶端膜移动（基础状态下峰值荧光为2.5±0.13微米，VIP、福斯可林和金雀异黄素刺激后分别为1.51±0.06微米、1.77±0.1微米和1.38±0.05微米；均P<0.001）。去除VIP后，氯离子分泌和顶端CFTR转运的增加均恢复到基础值。这些数据为完整上皮组织中CFTR转运的急性激素调节提供了首个定量形态学证据。
